PROJECT SUMMARY In normal cells, euchromatin and heterochromatin are functionally compartmentalized in three dimensions within the typically spheroid- or ovoid-shaped nuclei. Aged, progeroid, and cancer cells display characteristically irregular nuclei, with an accompanying loss of heterochromatin. Very little is known about how the chromatin compartments are independently anchored within the nucleus, or how such anchoring may be disrupted in ageing and cancer. The nuclear lamina is a filamentous protein meshwork located at the inner nuclear periphery that maintains nuclear shape and serves as the major organizing platform for heterochromatin. Here, the mechanism of heterochromatin organization will be investigated, and a novel hypothesis will be tested, that heterochromatin serves a reciprocal structural role in organizing the nuclear lamina. This hypothesis has been driven by our discovery of a human structural protein, PRR14, that functions as an essential bivalent tether to attach H3K9me3-modified, HP1-bound heterochromatin to the nuclear lamina. Interestingly, PRR14 is also required for the maintenance of nuclear shape. PRR14 participates in the earliest step of mitotic nuclear reassembly by first monovalently attaching to heterochromatin during chromosome separation in anaphase, and next completes bivalent attachment to the nuclear lamina during nuclear reassembly in telophase. We hypothesize that heterochromatin-bound PRR14 may thereby act as a platform to guide reassembly of the nuclear lamina meshwork. Defects in heterochromatin-nuclear lamina attachment in telophase could thus trigger, and perpetuate, nuclear lamina and chromatin organizational defects. Additional preliminary evidence indicates that mitotic disassembly and reassembly of PRR14 are phosphoregulated through the CDK1 kinase and PP2A phosphatase. Here we propose two Specific Aims that address fundamental hypotheses regarding heterochromatin organization and nuclear shape. In Aim 1, imaging methods will be used to test the hypothesis that heterochromatin-nuclear lamina tethering is important to organize the nuclear lamina during mitotic nuclear reassembly. Using a knockdown-add back approach, the ability of PRR14 mutants to support proper nuclear reassembly will be tested, including those mutations that specifically disable nuclear lamina or heterochromatin attachments. A second set of mutations is predicted to impact phosphoregulation of PRR14, and these will also be functionally characterized. In Aim 2, heterochromatin organization in mouse cells will be studied. Heterochromatin is organized differently in human and mouse cells, and we found that overexpression of the human PRR14 protein can trigger reorganization of mouse heterochromatin. We will perform a variety of comparative structure-function studies to understand the role of the mouse PRR14 protein and the H3K9me3 modification in heterochromatin organization. Successful completion of these aims will reveal fundamental mechanisms underlying nuclear organization, as well as identify potential origins of nuclear shape and chromatin defects in ageing and cancer.